Cherylyn A. Savary

Neuroendocrine Modulation of Signal Transducer and Activator of Transcription-3 in Ovarian Cancer

There is growing evidence that chronic stress and other behavioral conditions are associated with cancer pathogenesis and progression, but the mechanisms involved in this association are poorly understood. We examined the effects of two mediators of stress, norepinephrine and epinephrine, on the activation of signal transducer and activator of transcription-3 (STAT3), a transcription factor that contributes to many promalignant pathways. Exposure of ovarian cancer cell lines to increasing concentrations of norepinephrine or epinephrine showed that both independently increased levels of phosphorylated STAT3 in a dose-dependent fashion. Immunolocalization and ELISA of nuclear extracts confirmed increased nuclear STAT3 in response to norepinephrine. Activation of STAT3 was inhibited by blockade of the beta1- and beta2-adrenergic receptors with propranolol, and by blocking protein kinase A with KT5720, but not with the alpha receptor blockers prazosin (alpha1) and/or yohimbine (alpha2). Catecholamine-mediated STAT3 activation was not inhibited by pretreatment with an anti-interleukin 6 (IL-6) antibody or with small interfering RNA (siRNA)-mediated decrease in IL-6 or gp130. Regarding the effects of STAT3 activation, exposure to norepinephrine resulted in an increase in invasion and matrix metalloproteinase (MMP-2 and MMP-9) production. These effects were completely blocked by STAT3-targeting siRNA. In mice, treatment with liposome-incorporated siRNA directed against STAT3 significantly reduced isoproterenol-stimulated tumor growth. These studies show IL-6-independent activation of STAT3 by norepinephrine and epinephrine, proceeding through the beta1/beta2-adrenergic receptors and protein kinase A, resulting in increased matrix metalloproteinase production, invasion, and in vivo tumor growth, which can be ameliorated by the down-regulation of STAT3.

Prevention of rejection of allogeneic bone marrow transplants by NK 1.1 antiserum.

We have studied the effect of in vivo administration of NK 1.1 antiserum on two functions of natural killer (NK) cells: (A) in vitro cytotoxicity of B6 mice to T cell lymphoma, YAC-1, and (B) potential of B6 mice to reject allogeneic BALB/c bone marrow transplants. We demonstrated that a single i.v. injection of 0.4 ml of NK 1.1 antiserum significantly reduced in vitro NK cell cytotoxic potential and concomitantly prevented rejection of allogeneic bone marrow transplants. NK 1.1 antiserum was effective in diminishing both of these functions when injected 2, 6, or 24 hr before transplantation and NK cell assay, but it was ineffective when given 48 hr before transplantation or the NK cell test. As a specificity control, we investigated the effect of specific anti-T-cell-directed monoclonal antibodies, Thy 1.2 and Lyt 2.2, in the same systems. Neither of these antibodies exerted any effect on NK cell cytotoxicity to YAC-1 or rejection of allogeneic bone marrow transplants. These studies indicate that NK cells represent one of the major components of the mechanism of bone marrow graft rejection.

Aspergillus fumigatus Conidia Induce a Th1-Type Cytokine Response

The response of human peripheral blood mononuclear cells (MNC) to Aspergillus fumigatus in vitro was evaluated. In studies of the proliferative response of MNC from 18 healthy donors to heat-killed A. fumigatus conidia, 15 displayed a significant response, with a stimulation index (SI) between 4 and 193. In contrast, all donors displayed a positive response to Candida albicans blastoconidia (SI ranged from 10 to 224). Despite the variability in reactivity to A. fumigatus conidia, the response of a particular individual was stable when retested over periods of 1-2 weeks. Supernatant from cocultures of A. fumigatus conidia with MNC contained increased levels of interferon-gamma, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interleukin (IL)-2, compared with unstimulated cells, but not IL-10 or IL-4. In addition, A. fumigatus induced lymphocyte surface expression of adhesion/activation-associated molecules. These results suggest that lymphocytes may contribute to host defense against Aspergillus by generating a Th1-type response.

Multidimensional flow-cytometric analysis of dendritic cells in peripheral blood of normal donors and cancer patients

Abstract We studied the potential of multidimensional flow cytometry to evaluate the frequency and maturation/activation status of dendritic cells in minimally manipulated peripheral blood mononuclear cell preparations (i.e., only separated on Ficoll-Hypaque) of normal donors and cancer patients. A rare subset of HLA-DR+ leukocytes (less than 1% mononuclear cells) was detected in blood of normal donors that displayed all the features of dendritic cells: these cells had high forward-light-scatter characteristics and coexpressed CD4, CD86 and CD54 surface antigens, but lacked the lineage-associated surface markers of T cells, B cells, monocytes, granulocytes or NK i.e. they were CD3–, CD19–, CD20–, CD14–, CD11b–, CD16–, CD56–). These physical and phenotypic properties were virtually identical to those of immunomagnetically sorted leukocytes characterized as dendritic-cells on the basis of morphology, phenotype and high stimulatory activity in allogeneic mixed-lymphocyte cultures. Using this flow-cytometric approach we observed that the frequency of dendritic cell-like cells in peripheral blood mononuclear cell specimens of cancer patients receiving chemotherapy alone or those recovering from stem cell transplantation was significantly lower than that of normal individuals (mean ± SE: 0.36 ± 0.05%, 0.14 ± 0.06%, and 0.75 ± 0.04% respectively). Multidimensional flow-cytometric analysis of dendritic cells might represent an important new tool for assessing immunocompetence, and for monitoring the effects of therapeutic regimens on the immune system.

Dendritic cell-mediated stimulation of the in vitro lymphocyte response to Aspergillus.

Lymphocytes play a major role in host defense against Aspergillus, but little is known about the contribution of dendritic cells (DC) to antifungal immunity in humans. We have observed that DC derived from normal volunteers phagocytose heat-killed A. fumigatusconidia. Following 24 h of exposure to the fungus, DC displayed an increase in the mean fluorescence intensity of HLA-DR, CD80, and CD86, and an increase in the percentage of CD54+ cells. These DC also displayed increased production of IL-12. DC derived from CD34+ progenitors or monocytes stimulated autologous lymphocytes to proliferate and produce high levels of interferon-γ, but not interleukin-10, in response to fungal antigen. DC generated from CD34+ progenitors collected prior to autologous or allogeneic stem cell transplantation also partially restored the in vitro antifungal proliferative response of lymphocytes obtained from patients 1 month after transplantation. These results suggest that DC are important to host–response to A. fumigatus, and that ex vivo-generated DC might be useful in restoring or enhancing the antifungal immunity after hematopoietic stem cell transplantation. Bone Marrow Transplantation (2001) 27, 647–652.

IMPAIRMENT OF ANTIGEN-SPECIFIC CELLULAR IMMUNE RESPONSES UNDER SIMULATED MICROGRAVITY CONDITIONS

Abstract Microgravity has been implicated to play a role in the observed immune dysfunction of astronauts and cosmonauts after either short-term or long-term space travel. These reports, together with studies describing increased levels of microorganisms in the space cabin environment suggest potential risk for in-flight incidences of infectious diseases. In order to understand the mechanism underlying these immune defects, it is important to have a ground-based model that would reliably mimic the effects of microgravity on antigen-specific immune function. We tested the utility of the rotating wall vessel (RWV) technology developed at NASA as a model system because in the RWV the culture medium and the cells rotate synchronously with the vessel, thereby creating simulated microgravity conditions. We compared the RWV to the conventional tissue culture flask (T-flask), for culturing immune precursor cells with cytotoxic T lymphocyte (CTL) activity against synthetic viral peptides. We observed a significant loss of antigen-specific CTL activity in RWV cultures, but not in those from the T-flask, irrespective of the peptide immunogen used for inducing the primary immune response in different mouse strains. Loss of CTL activity in RWV cultures coincided with a significant reduction in CD8+ cells as well as CD4+ cells and DEC205+ dendritic cells, suggesting adverse effects of RWV culturing on both the effector and accessory cells for the loss of antigen-specific CTL function. These results provide a strong parallel to the reported defects in cell-mediated immunity during space travel and strongly support the utility of the RWV technology as an effective ground-based model for identifying key steps in immune cell dysfunction related to microgravity.

Tumoricidal effect of interleukin-2-activated killer cells in canines.

We investigated the effect of both partially purified (TCGF) and recombinant interleukin-2 (rIL-2) on the tumor-directed cytotoxic activity of canine peripheral blood mononuclear cells (PBMC) using three normal canines. No cytotoxic activity was displayed by unstimulated effector cells at 3 h of incubation; however, the cytotoxic effect was observed in a 16-h assay. PBMC of all canines displayed significant levels of lytic activity after stimulation for 4 to 7 days with both types of IL-2 against a variety of allogeneic and xenogeneic neoplastic cells in 3-h 51Cr release assay. The cytotoxic activity of cultured cells increased proportionally in the 16-h assays. Morphological examination of the May-Grünwald and Giemsa stained cytocentrifuged slides of cultured cells on each day of assay showed an increase in large granular lymphocytes (LGLs) beginning on day 4 and reaching a peak on day 7 of culture.

CHARACTERISTICS OF HUMAN DENDRITIC CELLS GENERATED IN A MICROGRAVITY ANALOG CULTURE SYSTEM

SummaryGeneration of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected, to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR+ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytoseAspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of Dc expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.

Depression of murine natural killer cell cytotoxicity by isobutyl nitrite

SummaryWe have investigated the effect of isobutyl nitrite on murine NK-cell antitumor-directed cytotoxicity. This agent has been suggested as one of the factors underlying immunodeficiency syndrome (AIDS) in man. We demonstrated that two injections, each of 0.25 ml isobutyl nitrite, resulted in significant depression of endogenous splenic and peripheral blood natural killer (NK) cell cytotoxicity against T-cell lymphoma, YAC-1. In addition to endogenous NK cells, activity of pyrimidinol-activated NK cells was also substantially depressed by this agent. The latter observation is of the utmost importance, since it suggests that the attempt to augment NK-cell activity (to promote resistance to infections and malignancies) could fail in patients with AIDS who are isobutyl nitrite users. Isobutyl nitrite was NK-cell-suppressive not only after in vivo administration but, most importantly, also after inhalation. This indicates that isobutyl nitrite, via its NK-cell suppressive effect, could contribute to immunodeficiency in AIDS. Studies on the mechanism of NK-cell depression by isobutyl nitrite demonstrated that the NK-cell tumor-binding properties as well as NK-cell cytotoxic potential were substantially depressed. Mixing experiments failed to reveal any regulation by suppressor cell activities. The results of these studies clearly indicate that isobutyl nitrite is an immunosuppressive agent and that its use should be avoided.

Cellular tumor vaccines administered after T cell-depleted allogeneic bone marrow transplantation induce effective anti-tumor immune responses.

In allogeneic hematopoietic stem cell (HSC) transplantation, graft vs. tumor (GVT) activity contributes to the cancer cure. It is closely associated with graft vs. host disease (GVHD), an immune response initiated by transplanted donor T-cell responses against host minor histocompatibility antigens (mHAgs). GVHD is prevented by T-cell depletion of the donor graft, but T-cell depletion also abrogates curative GVT. We wished to test the hypothesis that cellular tumor vaccines administered after T cell-depleted HSC transplant can induce significant GVT effects, despite the absence of transplanted mature donor T cells. In this investigation, a murine model of major histocompatibility complex (MHC)-matched, mHAg-mismatched allogeneic HSC transplant was studied. T cell-depleted or normal T cell-containing grafts were given to myeloablated recipients. Following reconstitution the recipients were vaccinated with tumor vaccines. GVT responses were measured in vitro by T-cell function assays and flow cytometry, and in vivo by tumor burden or survival. Post-transplant tumor vaccines induced effective anti-tumor responses in recipients of T cell-depleted transplants, producing cytolytic and cytokine responses, reduced tumor burden and prolonged survival. Recipients of T cell-depleted transplants that still have significant thymic function may be suitable subjects for post-transplant vaccine therapy.